Solid silica materials of various compositions and properties are available to facilitate purification, separation and detection of various molecules. In particular magnetic silica particles are used, because magnetic particles can be collected or retrieved by applying an external magnetic field to a vessel containing the particles. Unbound molecules and supernatant liquid can be separated from the particles or discarded, and the molecules bound to the particles may be retrieved e.g. by elution. Thus, magnetic particles provide relatively rapid, easy, and simple means to purify or separate molecules of interest such as nucleic acids from a liquid phase or a mixture of other molecules. Methods that are based on the use of silica particles are finding increasingly more applications in the purification or isolation of biomolecules, such as nucleic acids and proteins.
Therefore, there is a need for inexpensive and simple production methods for producing silica particles, in particular magnetic silica particles.
For the preparation of silica particles several methods are described in prior art:
DE 10 2006 054 173 describes a method for producing magnetic silica particles that is based on the use of magnetic core particles, a silica salt, a pH modifier, an organic pore forming agent and spray-drying. The produced silica particles exhibit good nucleic acid binding properties. However, spray-drying based methods as are described in said document are very costly due to the required equipment and the costs that accrue during operation of the spray dryer. Furthermore, spray dryer maintenance costs are often high, e.g. because the nozzles of the spray dryer can clog and other problems. Therefore, cost-efficient methods are desirous that allow the production of silica particles that are not based on spray-drying.
WO 1996/041811 describes the preparation of magnetic particles with an outer glass surface that are substantially pore free, or that have pores with less than 10 nm diameters. The particles are prepared by a gel-sol process by hydrolyzing tetraethyl orthosilicate in the acidic milieu. Once the gel is formed it is dried and densified by means of a thermal process to form glass.
WO 1998/031461 describes siliceous oxide-coated magnetic particles having a high resistance to leaching of the magnetic material on exposure to aqueous acidic environments. Disclosed is the preparation of the particles by deposition of siliceous oxide on core particles, wherein a siliceous source (preferably sodium silicate) and an acid (preferably a mineral acid) are added to the suspension containing the core particles. U.S. Pat. No. 6,027,945 describes the use of similar particles for the isolation of nucleic acids.
EP 0 757 106 describes magnetic silica particles comprising a superparamagnetic metal oxide. The particles are prepared by adding Fe3O4 particles to a tetraethoxysilane/alcohol solution. The deposition is started by adding a hydrolytic catalyst for tetraethoxysilane, which can be a weak or strong acid.
WO 2001/071732 discloses the preparation of porous magnetic silica particles by deposition of silicate onto magnetic core particles, wherein the source of silica can be a tetraalkoxysilane. The primary particles may form aggregates, thereby providing larger particles having a size of about 5 to 25 μm and having a favourable porous structure. The described particles have a good binding capacity for nucleic acids.
Thus, generally, silica is prepared industrially on large scale using two methods. One method is based on the hydrolysis of tetraalkoxysilane from an aqueous composition by the addition of a base or acid. The other method is based on acidifying a strong alkaline silicate composition. If these methods are performed in the presence of core particles, said core particles serve as nucleation sites for the deposited silica. When using magnetic core particles, such as for example iron oxide particles, magnetic silica particles are provided wherein the magnetic core particles are coated and thus are encapsulated by the deposited silica. Larger particles are usually formed by agglomeration of the coated core particles and potentially further coating of the formed aggregates.
When acidifying a silicate solution, the pH value is usually reduced from at least pH 12 to below 11. Thereby, the silicate becomes protonated, thereby providing silicic acid, which precipitates as SiO2×n H2O. It is mandatory to perform said process very slowly and in a large dilution, as otherwise the silicic acid precipitates spontaneously, thereby aggravating the production of defined, uniform particles. Still, when preparing magnetic silica particles by said method, there is a risk that aggregates having a very large particle diameter are prepared or that unmagnetic particles, so-called “fines” are provided, which are created by spontaneous hydrolysis and precipitation of silica, wherein said silica, however, is not deposited onto the magnetic core particles, thereby rendering unmagnetic silica particles. Therefore, the quality of the respectively produced silica particles is often not acceptable or a removal of deficient particles is required.
Thus, these prior art methods for preparing silica particles possess several disadvantages, such as the use of costly compounds (especially the tetraalkoxysilanes) or the disability to properly control the deposition process in order to generate particles of a relative uniform size. Spray drying based prior art methods for producing silica particles have the further disadvantage that the required equipment and the operation costs are expensive.
U.S. Pat. No. 3,678,144, U.S. Pat. No. 4,112,032 and U.S. Pat. No. 4,221,578 describe the production of silica bodies from silicate compositions. In the described methods, organic gelation agents such as formaldehyde, paraformaldehyde, formamide or glyoxal are added to the silicate solution which has a high pH value. Said organic gelation agents act as pH modulators. They uniformly dissolve in the silicate solution and react very slowly and uniformly throughout the solution, thereby continuously decreasing the pH value. Said decrease in the pH value of the reaction composition leads to a polymerization and thus precipitation of silica. Also Shoup in Coll. Interf. Sci. 1976, 3, 63-69 “Controlled pore silica bodies gelled from silica sol-alkali silicate mixtures” describes a respective method which involves the use of pH modulators to slowly reduce the pH value of the reaction composition, thereby precipitating silica in a controlled fashion. Here, a mixture of colloidal silica and alkali silicate is used in the reaction composition. Silica is precipitated by acidic hydrolysis of the silicate, the production of silicic acid, elimination of water and subsequent deposition of the silica onto the surface of colloidal silica particles, which provide as core particles a nucleation site for the precipitated silica. It is described that the colloidal silica remains as a stable dispersed phase in the reaction composition and serves as nucleation or growth site for polymerization of the molecular silicate. In this method, acidic hydrolysis of the silicate is achieved by adding a pH modulator. According to one embodiment, formamide is added as pH modulator, which is then hydrolysed to ammonium formate. The silica deposition on the surface of the colloidal core particles results in the formation of aggregates, wherein the primary core particles that are coated with the silica grow together thereby forming the silica body. The resulting pores are defined by the cavities between the particles which adhere due to the deposited silica. The shape of the formed silica body is determined in all said methods by the container in which the silicate solution is gelled. It is described that the formation of controlled pore bodies with narrow pore size distributions as described in the respective documents involve nucleation. Said silica production process which is based on the use of pH modulators is described in said documents only for the production of compact silica bodies which have the shape of the container the production process is performed in. The production of discrete silica particles is not described therein. Furthermore, the inventors found that it was not possible to produce discrete particles when following the respective teachings.
It is the object of the present invention to provide a method of preparing silica particles, in particular magnetic silica particles, which overcome the above mentioned disadvantages. In particular, it is the object of the present invention to provide a cost-efficient method which allows the production of silica particles, in particular magnetic silica particles, which have a high binding capacity for biomolecules, in particular nucleic acids.